黑曲霉W3350脯氨酰内肽酶在大肠杆菌中的表达

Expression of PEP of Aspergillus niger W3350 in E.coli

目的:构建pET-PEP重组原核表达载体,并对表达产物进行鉴定。方法:采用RT-PCR技术从黑曲霉(Aspergillus niger)W3350中扩增出脯氨酰内肽酶(PEP)的基因,插入表达载体pET-22b(+),经EcoRⅠ和HindⅢ双酶切和DNA序列测定验证,将正确的重组质粒转入大肠杆茵BL21(DE3)中,IPTG(终浓度1mmol/L)诱导获得表达。结果:表达产物进行超声破碎,经SDS-PAGE电泳检测,PEP分子质量大约为57kDa,与理论值相符,通过酶活方法测定脯氨酰内肽酶酶活为0.656U/ml,约是出发菌株的5倍。结论:首次成功构建表达载体pET-PEP并在原核细胞中表达,为进一步研究PEP的生物学功能奠定了基础。

Objective：To construct the prokaryotic expression vector of the pET-PEP,and identify the expressed production.Method：The PEP gene were amplified from Aspergillus niger W3350 cells by RT-PCR,and inserted into the prokaryotic expression vector pET-22b（＋） and verified by EcoRⅠ and HindⅢ digestion and DNA sequencing.The recombinant plasmid was expressed in E.coli BL21（DE3） by inducing of IPTG（1 mmol/L） and identified by SDS-PAGE.Result：After sonication,SDS-PAGE analysis indicated that the relative molecular mass（M）of the protein was about 57kDa,and it was equivalent to the expected value.the activity of PEP was 0.656U/ml by determination,and it is about as five times as the original strain.Conclusion：To construct the recombinant expression vector and to express the protein in E.coli successfully and to further the study of the biological function of PEP.